Translation, the process of protein synthesis, occurs via a universally conserved mechanism that is central to gene expression in all domains of life. Translation is highly regulated in human cells and the loss of translation control is a key determinant of cancerous cell growth. Protein synthesis in bacteria is targeted by a broad array of clinically-important antibiotics that are used to combat infectious disease. However, resistance to these compounds is increasingly widespread. The ribosome is the principal component of the cellular translation apparatus and is the integration point for regulation. In order to fill this knowledge gap, the molecular mechanism of ribosome function and translational fidelity will be investigated using state-of-the-art biophysical methods, including single-molecule Total Internal Reflection Fluorescence and zero-mode waveguide imaging technologies. Using these platforms, the first multidimensional, high-spatial and -temporal resolution distance measurements of distinct conformational degrees of freedom will be obtained during both elemental and processive protein synthesis reactions. Together with collaborative and complementary efforts in the areas of molecular dynamics simulations and cryo-electron microscopy, these investigations will reveal the order and timing of structural events in translation machinery and how they contribute to driving directional and high-fidelity protein synthesis. The long-term goal is to establish a quantitative framework that relates the microscopic rate constants of conformational events in the ribosome to global protein synthesis. This will shed new light on the rate-determining structural events in the process as well as the molecular basis of translation fidelity, and will provide insights critical to understanding cellular mechanisms of ribosome regulation and the action of clinically-relevant small molecule effectors of translation. A synthesis of the results obtained will provide novel information about precisely focused, dynamic structural processes underpinning the translation mechanism and a platform for exploring how specific events that occur during the reaction coordinate may be targeted for therapeutic purpose. PUBLIC HEALTH RELEVANCE: The focus of the proposed research is to apply state-of-the-art imaging technologies to the study of translational control of gene expression. This multistep and highly regulated process is central to growth, differentiation and tumorigenesis and compounds targeting translation are central components of the arsenal of therapies for the treatment of disease.